Method for investigating the fate of a test compound or the stateof a biological system by means of NMR of hyperpolarised NMR active nuclei

ABSTRACT

The invention is concerned with Nuclear Magnetic Resonance (NMR) spectroscopy and Magnetic Resonance Imaging (MRI), particularly NMR spectroscopy. It provides hyperpolarization methods offering enhanced sensitivity of detection over conventional NMR for studying the fate of a test compound in a biological system. The methods are particularly suitable for studying metabolism and toxicity of drugs. The resulting NMR sensitivity increase is advantageous in two key aspects of NMR detection: test compounds can be detected at lower concentrations and substantial time saving can be achieved in cases where extensive averaging is conventionally employed to increase the signal to noise ratio of the corresponding NMR spectra. The methods can be used for studios that were not practical or not possible using conventional NMR.

Technical Field

This invention is concerned with Nuclear Magnetic Resonance (NMR)spectroscopy and Magnetic Resonance Imaging, particularly NMRspectroscopy. The spectra of NMR active nuclei vary depending on theirenvironment. The present invention provides a method for obtaininginformation regarding the fate of a test compound, which may beexogenous, e.g. a drug, or an endogenous native compound, in abiological system by enhancing the nuclear polarisation of NMR activenuclei of the test compound (hereinafter termed ‘hyperpolarisation’)prior to NMR or MRI analysis. The invention also provides a method forcarrying out NMR pattern profiling to obtain information on the statusof a biological system.

Definitions

The term ‘test compound’ as used hereinafter, refers to a compound thatmay be exogenous or endogenous to the biological system in which it isto be studied and that is of physiological interest, e.g. a potentialdrug substance, which is not a noble gas nor a so-called Overhauser MRI(OMRI) contrast agent or other MR Imaging agent. A test compound mustcontain at least one NMR active nuclei, i.e. a nuclei with non-zeronuclear spin.

The term ‘fate’ as used hereinafter when applied to a test compoundencompasses metabolism, absorption, distribution and excretion in abiological system.

The term ‘biological system’ as used hereinafter encompasses wholeanimals, plants, micro-organisms, isolated organs and tissues, isolatedcells or cultured cells isolated sub-cellular organelles and expressedand/or reconstituted enzyme systems. Samples that may be extracted frombiological systems such as whole animals, plants, isolated organs ortissues, include tissue or cell samples, faeces, body fluids includingbut not limited to blood, lymph, urine, semen, breast milk,cerebro-spinal fluid, sweat, lachrymal or parotid secretions or lavage.

Background and Brief Summary of Invention

Due to the genomic revolution, combinatorial chemical libraries and highthroughput screening, an exponentially increasing number of new chemicalentities is now entering or is already in the trial phases requiredprior to marketing as new drugs. This rapid evolution of potentiallybeneficial drugs has led to an increased pressure on both the efficacyand safety evaluation processes. There is an on-going intensive searchfor new technologies that may optimise the efficiency of suchevaluations.

Pharmacokinetic and toxicology testing have two key requirements, whichare the identification of metabolites formed from the parent compoundand assessment of the toxicity of both the parent compound and itsmetabolites. During pre-clinical tests and the clinical trial phases ofdrug development, it is essential to investigate (bydetection/monitoring) whether trial drugs themselves or theirmetabolites give rise to adverse reactions in in vitro test systems,animals, healthy volunteers or patients. It is also necessary toascertain whether potentially undesirable and even dangerous reactionsare related to the concentration or distribution of the drug or one ormore of its metabolites in the body. In addition such evaluations may beconducted in selected patients in order to determine whether particulargroups of patients with, for instance, identified defects in one or moredrug metabolising enzymes (which may represent a very small minority ofthe pre-clinical and clinical trial populations) are at an increasedrisk of developing adverse drug reactions. An important aspect of anysuch investigations is to determine the fate of a drug substance once ithas been administered, i.e. its absorption, tissue distribution, rateand site(s) of metabolism, characterisation of structure and relativeabundance of metabolites and routes of excretion. There is a need fornew methods for studying the fate of a test compound.

One of the methods that can be used to study the fate of a test compoundin a biological system is to identify the structures of its metabolites.Current techniques for identifying the structure of metabolites relyheavily on mass spectroscopy (MS) in combination with liquidchromatography. However, mass spectroscopy is, on its own, often notable to characterise the structure of metabolites fully andunambiguously. Data derived from NMR spectroscopy are oftencomplementary to that obtained from MS and when used in combinationthese techniques may allow the structure of metabolites to bedetermined. Unfortunately, currently NMR is relatively insensitive. Inmany cases, the relatively low sensitivity of NMR creates fundamentalproblems that affect the acquisition time needed in order to achieve adesired signal and the lower limit of detection (LOD) of analyte at adefined signal:noise ratio (e.g. 3:1).

In practical terms, the poor sensitivity of current NMR techniqueslimits their application in absorption, distribution, metabolism andexcretion (ADME) studies. During the early stages of drug development,the supply of candidate drug, and hence its metabolites, is limited andthere is often not enough material available for analysis by NMR. Inaddition, the concentration of metabolites produced by in vitro and invivo screens is low and often well below the level needed for analysisby NMR. It is not advisable to increase dosing because the routes ofmetabolism may change under such non-physiological conditions and themetabolites formed will be non-representative of these produced understandard patient treatment regimes.

At present, it is necessary to scale up the testing procedure and toconcentrate the metabolites from large volumes of biological fluids(e.g. cell culture superntants, organ perfusates, plasma, bile, urineetc.) in order to characterise the new candidate drugs by NMR. This isvery time consuming so, in practice, it often does not take place untillate in the drug development phase. This leads to many of the costlylate phase candidate drug failures. Ideally, NMR needs to beincorporated as a routine analytical tool alongside MS as early aspossible in the evaluation of the ADME characteristics of new chemicalentities. However, this is not feasible with the sensitivity ofconventional NMR techniques. The present invention using hyperpolarisedNMR active nuclei addresses many of the aforementioned limitations andhence offers many advantages compared to conventional NMR techniques, aswill be discussed below.

In addition to ADME applications, toxicity (Tox) evaluations are alsocentral to the drug approval process. The current situation with regardto Tox testing is arguably even more problematic than ADME. Manycandidate drugs are found to exhibit unacceptable toxicity late inclinical trials, and even occasionally post launch. It is widelyaccepted within the pharmaceutical industry that current pre-clinicaltoxicological screens are inadequate. Current in vitro screens arepoorly predictive of the in vivo situation. Consequently, the toxicityof new candidate drugs must be evaluated thoroughly in two animalspecies before large scale testing in humans. This is costly and timeconsuming. Moreover, results from animal testing are not alwayspredictive for humans. There is an urgent requirement for improvedtoxicity screening procedures.

Bioanalytical approaches for evaluating drug efficacy and safetycurrently include measurements of responses of living systems to drugcandidates either at the genetic level or at the level of expression ofcellular proteins, using so-called genomic and proteomic methodsrespectively. However, since both methods ignore the dynamic metabolicstatus of the whole cell, tissue or organism, even in combinationgenomics and proteomics may not provide sufficient information aboutintegrated cellular function in living systems to assess accurately thefate and toxicological profile of a drug candidate. A high-resolution ¹HNMR-based approach has been suggested (Xenobiotica, 1999, vol. 29,1181-1189, J. K. Nicholson et al) and has been termed metabonomics.Metabonomics is defined as the quantitative measurement of the dynamicmultiparametric metabolic response of living systems topathophysiological stimuli or genetic modification. It is anticipatedthat such analyses will highlight patterns of variations of endogenouscompounds produced in response to known toxins. This should enable thetoxicity of new candidate drugs to be predicted by comparison. Themethods according to the present invention using hyperpolarised NMRshould enable improvements in metabonomic analyses of the effect of newcandidate drugs on the distribution, metabolism and excretion ofendogenous compounds in comparison to currently employed techniques.Hyperpolarised NMR will enable the utilisation of ¹³C NMR for thesesorts of studies. Currently, only the use of ¹H NMR is practicable (dueto insensitivity of detection for ¹³C using conventional NMR) and theinformation content of the analysis is limited by the chemical shiftrange of ¹H. In comparison, ¹³C NMR offers a much wider range ofchemical shifts. The improvements obtained by the methods of the presentinvention may be in terms of speed and sensitivity and any combinationthereof.

NMR pattern profiling is a technique that is used to acquire informationabout the status of a biological system (J Pharm Biomed Anal March 1995,13(3): 205-11, Anthony M L et al; Mol Pharmacol July 1994; 46(1):199-211, Anthony M L et al; Naturwissenschaften January 1975;62(1):10-4, Kowalski B R and Bender C F). Typically an NMR pattern,which may be a spectrum or an image, from a system that has beensubjected to some kind of perturbation in its state is compared with theNMR pattern from the same type of system in its usual state. Changes inthe pattern can then be correlated with the change in state of thesystem. The change in state of the system may be, for example, exposureto a drug substance, change in environment or a disease, or a change instage of development of the system. Profiling can be used to compare twosystems to determine whether they are in the same or in differentstates. Once information is available on the pattern exhibited by a typeof system in a variety of states, profiling can be used to determine thestate of a test system of that type by comparing the pattern exhibitedby the test system with the known patterns for that type of system. Theinformation is conveniently stored electronically and algorithmicanalyses can be used to compare the pattern for the test system with theknown patterns. The algorithmic analyses are suitably carried out usinga computer and appropriate software. NMR pattern profiling ispotentially a powerful technique for acquiring a plethora of informationabout a system even when specific entities, for example metabolites,cannot unambiguously be identified individually. However, its usefulnesshas been limited by the inability of current technology to detect andresolve differences in individual spectra due to the relatively lowsensitivity of the NMR technique. The present invention usinghyperpolarised NMR active nuclei addresses this limitation and therebypotentially enables NMR pattern profiling to be used to obtain moreinformation than is available using conventional NMR techniquesregarding, for example, a system's health, function and metabolicstatus, and the mechanisms occurring within the system.

The present invention is not limited to any specific method forpolarising NMR active nuclei. Such polarisation may be achieved by manydifferent ways, for example, by polarisation transfer from a noble gas,or by one of the ‘Brute force’ (WO 99/35508, Nycomed Imaging AS), DNP(WO 98/58272, Nycomed Imaging AS) and para hydrogen (p-H₂) methods (WO99/24080, Nycomed Imaging AS) as explained below.

Noble gas isotopes having non-zero nuclear spin can be hyperpolarised,i.e. have their polarisation enhanced over the equilibrium polarisation,e.g. by the use of circularly polarised light. Preferred techniques forhyperpolarisation include spin exchange with an optically pumped alkalimetal vapour and metastability exchange. Noble gases to which thistechnique can be applied include ³He and ¹²⁹Xe. The enhanced nuclearpolarisation of a noble gas can be transferred to another NMR activespecies in close proximity by spin-spin interaction. WO 97/37239(Lawrence Berkeley National Laboratory) describes methods fortransferring nuclear polarisation from a hyperpolarised noble gas tonuclear spins on a target compound, leading to an enhancement of thecorresponding NMR or MRI signals. WO 98/30918 (Nycomed Imaging AS)relates to ex-vivo dynamic nuclear polarisation (DNP) or NuclearOverhauser Effect (NOE) cross-polarisation from a hyperpolarised gas toan MRI agent where the gas is separated from the MRI agent prior toadministration to the body.

Although the NMR spectroscopy or imaging method of the present inventionprovides similar types of information about the fate of a test compoundas conventional NMR or MRI, it offers potential advantages. Among theseadvantages are: a) increased sensitivity of analysis; and b) increasedspeed of acquisition of NMR spectra (or images). The analysis ofdrugs/metabolites or physiological compounds containing an NMR activenuclei may provide additional information previously only supplied bystudying corresponding ¹⁴C-labelled compounds, whilst being free fromthe problems associated with radioactive isotopes.

Furthermore, in comparison to studies using fluorescent reagents andrelated labelling technology, for example, the current invention doesnot require the synthesis of an adduct comprising a reporter, e.g. afluor, and the test compound in order to enable detection. Therefore thepresent invention offers the following advantages over conventionalfluorescence based detection systems:

-   -   1. There is no alteration in the chemical structure of novel        drugs or physiological compounds. There is always a disadvantage        with techniques such as fluorescent methods in that the        additional chemical component may influence the measurement.        Specifically, the fluorescent label may be of a significant size        when compared with the test compound and sometimes as large or        larger than the test compound. Consequently the fate of the        labelled test compound may be quite different from the        non-labelled compound.    -   2. Fluorescence measurement may not be specific, due for example        to dye leakage, dye compartimentalisation, quenching of signal        and autofluorescence.

Similarly, although the NMR pattern profiling method of the presentinvention provides similar types of information about the state of asystem when compared to existing NMR pattern profiling methods, it alsooffers potential advantages. Among those possible advantages are: a)increased sensitivity of analysis; and b) increased speed of acquisitionof NMR spectra (or images). Increased detail in pattern profiles, may berealised as a consequence of increased sensitivity such that featuresbecome visible within spectra that would not be discernible from noiseunder conventional NMR. The sensitivity increase enables utilisation of¹³C NMR as well as ¹H NMR which collectively would provide additionalinformation relative to ¹H NMR profiles alone. Changes in patterns thatare not visible in pattern profiles obtained using conventional NMRtechniques may be observed using the method according to the presentinvention. These small changes may be particularly important for examplewhen studying toxicity or carrying out quality assurance testing on cellcultures.

BRIEF DESCRIPTION OF DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood when the following detaileddescription is read with reference to the accompanying drawings in whichlike characters represent like parts throughout the drawings, wherein:

FIG. 1 illustrates the NMR spectrum of (13C carboxyl) Benzoic acidisolated from rat urin wherein the prominent peak from the leaveled¹³COOH site of benzoic acid is clearly visible at 176.0 ppm and whereinthe prominent signals at around 71.9ppm and around 62.3 ppm areidentified as solvent peaks from glycerol.

FIG. 2 illustrates an enhanced NMR spectrum of a sample isolated fromrat uring after administration of (13C-carboxyl) Benzoic acid.

FIG. 3 illustrates an enhanced NMR spectrum of a sample isolated fromrate bile after administration of (U-13C) paracetamol.

DETAILED DESCRIPTION

One aspect of the present invention concerns a method for monitoring anyaspect of the fate of a test compound, including metabolism, whichmethod comprises polarising one or more NMR active nuclei in the testcompound and detecting changes in the spectra of the nuclei. The testcompound may be an exogenous compound such as a drug or an endogenous‘native’ substance. The changes may be detected continuously or as aseries of discrete measurements or as a single measurement. Bothquantitative and qualitative measurements are included, especially thedynamic clearance pattern of any metabolites using samples of e.g.exhaled respiratory gases, blood, blood plasma, urine or other bodyfluids. Suitable nuclei are those with non-zero nuclear spin. PreferredNMR active nuclei are ¹³C, ¹⁵N, ³¹P, ¹⁹F and/or ¹H, ¹³C is particularlypreferred

Where the test compound is a drug, and isotopic enrichment isappropriate to facilitate detection it is preferred to use stable,non-radioactive isotopes that have substantially no effect on thetherapeutic efficacy of the drug, such as ¹³C and ¹⁵N. Alternatively,nuclear species occurring at high natural abundance such as ³¹P, ¹⁹Fand/or ¹H, can be detected according to the methods of the invention.

Where the test compound is exogenous, e.g. a drug, it may be polarisedbefore administration to the system in which its fate is to be studied.Alternatively, for endogenous and exogenous test compounds, the wholesystem or samples extracted from the system may be subjected to anappropriate hyperpolarisation technique at various times.

Thus, in a first aspect the present invention provides a method forinvestigating the fate of a test compound containing at least one NMRactive nuclei said method comprising:

-   -   administering the test compound to a biological system;    -   hyperpolarising the NMR active nuclei in the system-or in a        sample extracted from the system; and    -   analysing the hyperpolarised system or one or more samples        extracted from the system by NMR spectroscopy and/or NMR        imaging.

NMR spectroscopy is the preferred method of analysis, particularly wheresamples extracted from the system are to be analysed.

Suitably, where the system is an animal or perfused organ, samples (e.g.biopsy, necropsy or fluid extract) will be taken and thenhyperpolarised. For example, blood or urine samples may be taken. Thesamples may be purified prior to NMR spectroscopy, but this is notalways necessary. An important advantage of the methods according to thepresent invention is that, unlike with prior art methods, spectroscopycan be carried out directly on the crude biological sample without theneed for fractionation, purification or concentration steps.

This method is particularly suitable for dynamic studies as samples maybe taken at time intervals, hyperpolarised, and then NMR spectra of thevarious samples can be compared to show changes over time.Hyperpolarisation may be effected by means of a polarising agent as asingle transfer, continuous transfer or intermittent transfer. Anappropriate method for hyperpolarisation will be selected depending onthe nature of the system or sample.

The test compound is preferably exogenous to the biological system inwhich its fate is to be studied, e.g. a drug or drug candidate.

In another aspect, the invention provides a method for investigating thefate of a test compound containing at least one NMR active nuclei, whichmethod comprises:

-   -   hyperpolarising the NMR active nuclei in the compound;    -   administering the hyperpolarised compound to a biological        system; and    -   analysing the system or samples extracted from the system by NMR        spectroscopy and/or NMR imaging.

An appropriate method of hyperpolarisation will be selected depending onthe nature of the test compound.

NMR spectroscopy is the preferred method of analysis, particularly wheresamples extracted from the system are to be analysed. The test compoundis preferably exogenous to the biological system in which its fate is tobe studied, e.g. a drug or drug candidate. Again, the samples may besubjected to preliminary steps such as fractionation, purification orconcentration prior to spectroscopy, but the fact that this is notalways necessary is an advantage of this method.

Suitable NMR active nuclei for use in test compounds for the methodsaccording to the first and second aspects include ¹³C, ¹⁵N, ³¹P, ¹⁹Fand/or ¹H. ¹³C and ¹⁵N are particularly suitable. ¹³C is the mostpreferred.

Although it may be possible to employ the methods of the invention withtest compounds containing a natural abundance of the NMR active nuclei,where the test compound is exogenous, it is preferably enriched with NMRactive nuclei before administration to the system. This may includeeither selective enrichments of one or more sites, or uniform enrichmentof all sites. Enrichment can be achieved by chemical synthesis orbiological labelling. Preferably, a test compound for use in a methodaccording to the invention is an organic compound comprising anartificially enriched abundance of, for example, ¹³C, either generallyor at least in one specific position, at an abundance of at least 5%,suitably at least 10%, more suitably at least 50%, preferably at least75%, more preferably at least 90% and ideally at approaching 100%.

The present invention also covers the use of test compounds comprisingan artificially-enriched abundance of ¹⁵N of at least 1%, suitably atleast 5% more suitably at least 10%, preferably at least 50% and morepreferably at least 75% or more, and ideally at approaching 100%.

Enrichment of more than one nuclear species, e.g. ¹³C and ¹⁵N may beperformed in the same test compound.

Although it might be expected that, because different ¹³C centres in auniformly enriched test compound relax at very different rates, verydifferent signal intensities would be found in any NMR spectra produced,surprisingly peaks have been observed even for ¹³C centres that wereexpected to relax too rapidly to appear in spectra. Thus, a peak foreach carbon centre can be observed in spectra produced according to apreferred embodiment of the invention wherein the NMR nuclei are ¹³C andanalysis is by spectroscopy.

The degree of hyperpolarisation of the NMR active nuclei or nucleiaccording to this invention can be measured by its enhancement factorcompared to thermal equilibrium at spectrometer field and temperature.Suitably the enhancement factor is at least 10, preferably it is atleast 50 and more preferably it is at least 100. However test methodswhere even smaller enhancements are achieved may still be performedusefully due to the shorter time needed for the total measurementcompared with conventional methods. If the enhancement is reproducibleand the polarisation/NMR measurement can be repeated, the signal tonoise ratio of an NMR signal can be improved. In such a case, theminimum NMR enhancement factor required depends on: a) the polarisationtechnique and b) the concentration of the test compound. The enhancementhas to be large enough so that the NMR signal from the test compound canbe detected. In this context, it is clear that an enhancement of 10 orless than 10 that it is achievable in a multi-shot experiment may bevery useful due to the time saved in data acquisition compared withconventional NMR.

The analysis steps of the above mentioned methods may be carried out bycontinuous monitoring or as a single discrete measurement or as a seriesof discrete measurements that may be carried out at suitable intervalsover time. Thus, changes in the spectra or images can be monitored overtime and correlated with dynamic events. Such dynamic events may includemetabolic events, changes in distribution, progress in absorption, andprogress in excretion of the test compound The aforementioned methodsmay be used to monitor the dynamic fate of any test compound as well asendogenous metabolites or metabolites of exogenous test compound usingsamples from e.g. blood, urine or other body fluids.

When the fate to be studied relates to metabolism, NMR spectroscopyrather than MRI should be used as the method of analysis. It may bepossible, by carrying out the analysis over time, to identify many andpreferably all known changes in metabolism or appearance of individualmetabolites of the test compound. It should be possible to assignspecific peaks in the spectrum to known metabolites. The increasedsensitivity of the technique may result in additional peaks (comparedwith spectra obtained using conventional NMR) due to previouslyunrecognised minor products of metabolism appearing in the spectrum.This is important because even tiny amounts of toxic metabolites cancause a drug candidate to exhibit damaging side-effects. This methodwill thus be a very useful tool to evaluate the metabolic/toxicitypattern for a drug or other substances as well as producing mechanisticinformation.

Metabolic studies can be carried out in whole animals, perfused organs;tissue or cell cultures or in test tube systems utilising, for example,microsomal preparations or other sub cellular fractions such as S9 mix(which contains both phase one and phase two metabolising enzymes).Where whole animals or organs are used, it is preferred to employ themethod according to the first aspect of the invention and tohyperpolarise samples extracted from the animal or organ. Blood andurine samples are particularly suitable. Studying urine samples has theadvantage of enabling cumulative effects to be observed.

The methods will be particularly useful when some of the metabolites arenot previously known. The chemical shift from the polarised NMR activenuclei may help to identify the nature of the new metabolites.

In addition, the methods may also be useful even if it is not possibleto identify the different metabolites unequivocally because in somesituations the dynamic clearance pattern from unknown metabolites mayalso have a significant value.

When the fate of the test compound to be studied is absorption, thesystem is conveniently a whole animal, perfused organ, tissue or cellsystem. Whole animals and perfused organs, especially whole animals, areparticularly suitable. Typically, samples are extracted from differentlocations in the system and analysed by NMR spectroscopy. Alternatively,the whole system can be analysed by NMR imaging. In one embodiment, thetest compound is hyperpolarised and then administered to the human oranimal body by inhalation. Absorption of the test compound via the lungsis monitored by NMR imaging. In another embodiment, the test compound ishyperpolarised, then administered to the human or animal body byintravenous injection and absorption from the bloodstream is observed byNMR imaging.

If it is desired to investigate the distribution of a test compound, itis preferred to hyperpolarise the compound and then administer it to awhole animal or human body. The compound may be administered byinhalation, or alternatively it can be administered intravenously. NMRimaging is preferably used to monitor the distribution of the testcompound. Suitably, imaging can be carried out continuously.Alternatively, a series of discrete images can be produced over time.NMR imaging can be carried out on the whole human or animal body.Alternatively, solid sections can be taken from an animal that has beenkilled at a known time interval from administration of the test compoundand these sections can be imaged. In this case, the solid sections arehyperpolarised before imaging.

If the fate of the test compound to be investigated is excretion, it ispreferred to administer the test compound to a whole animal and then toextract samples of, for example, bile, saliva, faeces, urine or exhaledair. These samples are hyperpolarised prior to NMR analysis. NMRspectroscopy should be employed in studies of this type.

In a third aspect, the present invention provides a method forinvestigating the state of a biological system containing at least oneNMR active nuclei, which method comprises:

-   -   hyperpolarising the NMR active nuclei; and    -   analysing the system or samples extracted from the system by NMR        spectroscopy and/or NMR aging to generate a NMR pattern of the        system.

The hyperpolarisation step may be carried out on the whole system or ona sample extracted from the system.

In one preferred embodiment, the following additional steps are carriedout:

-   -   subjecting the system to a change in its state;    -   hyperpolarising the NMR nuclei;    -   analysing the system or samples extracted from the system in its        changed state by NMR spectroscopy and/or NMR imaging to generate        an NMR pattern of the system in its changed state;    -   comparing the NMR patterns of the system and the system in its        changed state and identifying any changes in the NMR pattern.

The changes in NMR patterns identified in the final step can becorrelated with the change in state of the system.

Alternatively, two or more systems of the same type may be studied. Thefirst, or test, system is subjected to a change in its state, while thesecond, or control system is not. The hyperpolarisation and analysissteps are carried out on each system and the NMR patterns are comparedand any differences between patterns for the test system and the controlsystem are identified.

Thus, in the fourth aspect, the present invention provides a method forinvestigating the state of a biological system containing at lease oneNMR active nuclei which comprises:

-   -   subjecting the system to a change in its state;    -   hyperpolarising the NMR nuclei;    -   analysing the system or samples extracted from the system by NMR        spectroscopy or NMR imaging to generate an NMR pattern of the        test system;    -   comparing the pattern with a pattern obtained from a control        system that was not subjected to a change in its state prior to        hyperpolarisation and analysis; and    -   identifying any differences between the pattern from the test        system and the pattern from the control system.

Several test systems can be subjected to the same or different changesin state and their NMR patterns can be compared with the pattern from asingle control system.

The state of the system may be changed by external or internalinfluences. Examples of external influences include exposure to anexogenous substance, such as a drug or other type of test compound, oralterations in the environment of the system, e.g. changes intemperature or pH. An example of an internal influence is thedevelopment of the system over time, e.g. cell growth anddifferentiation.

Suitably the NMR patterns are stored electronically, for example in adatabase. Algorithmic analysis is conveniently used to carry out thecomparison step, typically by employing a computer with appropriatesoftware.

NMR spectroscopy is the preferred method of analysis. The NMR activenuclei is suitably a nuclei with non-zero nuclear spin. Preferred activenuclei are ¹³C, ¹⁵N, ³¹P, ¹⁹F and/or ¹H. ¹³C is particularly preferred.

The methods can be repeated to acquire NMR patterns for a system in anumber of different states. This information is conveniently storedelectronically, for example in a database. When one or more NMR patternsare available for a system in a known state or states, these can becompared with the NMR pattern for a system of the same type in anunknown state. If the NMR patterns are substantially similar or the samefor the system in a known state and the system in an unknown state, theunknown state will be the same or similar to the known state. Normally,the more NMR patterns available for a particular type of system in avariety of states, the more likely it is to find a pattern substantiallysimilar or the same as the pattern for a test system of that type in anunknown state.

Thus, in a fifth aspect, the invention provides a method forinvestigating the state of a test biological system containing at leastone NMR active nuclei, which method comprises:

-   -   hyperpolarising the NMR active nuclei;    -   analysing the test system or samples extracted from the test        system by NMR spectroscopy and/or NMR imaging to generate an NMR        pattern for the test system;    -   comparing the NMR pattern for the test system with the NMR        pattern for at least one other system of the same type as the        test system, said other system being in a known state when its        pattern was generated;    -   determining the state of the test system.

The hyperpolarisation step may be-carried out on the system or on asample extracted from the system.

Conveniently, the comparison step is carried out by algorithmicanalysis, for example by using a computer with suitable software.Suitably, the NMR pattern for the test system is compared with severalother NMR patterns and preferably with a significant number of other NMRpatterns stored electronically, for example in a database.

The accuracy of this method will depend, amongst other factors, on thesimilarity of the test system with the systems of the same type forwhich NMR patterns are available. Thus, more accurate results will beobtained regarding the state of isolated test cells where the cells arederived from the same lineage, tissue and species as the cell culturesfor which patterns are known, than if the test cells are derived from adifferent cell lineage, tissue or species. The method is particularlyuseful for quality assurance testing of systems that are intended to bethe same, e.g. cell cultures, wherein small but physiologicallysignificant differences can be detected.

The methods of the third, fourth and fifth aspects are useful forinvestigating responses of a biological system to a compound, e.g. adrug, about which relatively little is known. Useful data about theeffects of the compound can be obtained by comparing the NMR pattern ofa system exposed to it in various quantities with the NMR patterns forthe system when exposed to other compounds. For example, the patternsobserved for cells derived from human liver when exposed to a testcompound can be compared with NMR patterns for the same cells whenexposed to substances with known effects on the liver. If the patternfor cells exposed to the test compound is similar to the pattern forcells exposed to a compound that is known to have liver toxicity, it islikely that the test compound will also exhibitliver toxicity. This isparticularly useful when a baseline of pre and post testing strategiescan be established using the same system. NMR profiling can also provideimportant structural information about unknown compounds.

The profiling methods of the third, fourth and fifth aspects areparticularly suitable for studying plants.

A polarised noble gas, preferably ³He or ¹²⁹Xe, or a mixture of suchgases, may be used according to the present invention to effect nuclearpolarisation of the test compound or system comprising at least one NMRactive nuclei. The hyperpolarisation may also be achieved by using anartificially enriched hyperpolarised noble gas, preferably ³He or ¹²⁹Xe.The hyperpolarised gas may be in the gas phase, it may be dissolved in aliquid, or the liquefied hyperpolarised gas itself may serve as asolvent. Alternatively, the gas may be condensed onto a cooled solidsurface and used in this form, or allowed to sublime. Either of thesemethods may allow the necessary intimate mixing of the polarised gaswith the target to occur. In some cases, liposomes or microbubbles mayencapsulate the hyperpolarised noble gas.

In a further embodiment, the present invention provides a method whereinthe polarisation may be imparted to atoms of significance in the testcompound or system (e.g. ¹³C, ¹⁵N, ³¹P, ²⁹Si, ¹⁹F and ¹H isotopes) bythermodynamic equilibration at a very low temperature and high field.Hyperpolarisation compared to the operating field and temperature of theNMR spectrometer is effected by use of a very high field and very lowtemperature (Brute force). The magnetic field strength used should be ashigh as possible, suitably higher than 1T, preferably higher than 5T,more preferably 15T or more and especially preferably 20T or more. Thetemperature should be very low e.g. 4.2K or less, preferably 1.5K orless, more preferably 1.0K or less, especially preferably 100 mK orless. It will be appreciated that this embodiment is not suitable forpolarising a viable biological system where that system is a wholeanimal, isolated organ or tissue or cultured cells.

In a further embodiment, the present invention provides a method forpolarisation transfer using the DNP method effected by a DNP agent, toeffect nuclear polarisation of the test compound or system comprising atleast one NMR active nuclei. DNP mechanisms include the Overhausereffect, the so-called solid effect and the thermal mixing effect.

Most known paramagnetic compounds may be used as a “DNP agent” in thisembodiment of the invention, e.g. transition metals such as chromium (V)ions, magnesium (II) ions, organic free radicals such as nitroxideradicals and trityl radicals (WO 98/58272) or other particles havingassociated free electrons. Where the DNP agent is a paramagnetic freeradical, the radical may be conveniently prepared in situ from a stableradical precursor by a radical-generating step shortly before thepolarisation, or alternatively by the use of ionising radiation. Duringthe DNP process, energy, normally in the form of microwave radiation, isprovided, which will initially excite the paramagnetic species. Upondecay to the ground state, there is a transfer of polarisation to a NMRactive nuclei of the target material. The method may utilise a moderateor high magnetic field and very low temperature, e.g. by carrying outthe DNP process in liquid helium and a magnetic field of about 1T orabove. Alternatively, a moderate magnetic field and any temperature atwhich sufficient NMR enhancement is achieved in order to enable thedesired studies to be carried out may be employed. The method may becarried out by using a first magnet for providing the polarisingmagnetic field and a second magnet for providing the primary field forMR spectroscopy/imaging. It will be appreciated that, as in the Bruteforce method described above, that this embodiment is not suitable forpolarising a viable biological system where that system is a wholeanimal, isolated organ, or tissue or cultured cells if high fields andlow temperatures are used.

It might be expected that the presence of a paramagnetic radical wouldcause line-broadening and susceptibility shifts in NMR spectra producedin analysing the sample. Pleasingly, this does not occur in theexperiments carried out to date. This good result may be explained bythe low relaxivity of the radical used and its low concentration in thefinal sample. Therefore it is preferred to use a DNP radical with lowrelaxitivity in those embodiments of the invention where a DNP radicalis required.

In a further embodiment, the present invention provides a para hydrogeninduced polarisation method. Hydrogen molecules exist in two differentforms, para hydrogen (p-H₂) where the nuclear spins are anti paralleland out of phase (singlet state) and ortho hydrogen (0-H₂) where thespins are parallel or anti parallel and in phase (triplet state). Atroom temperature, the two forms exist in equilibrium with a 1:3 ratio ofpara:ortho hydrogen. However, preparation of para hydrogen enrichedhydrogen can be carried out a low temperature, 160K or less, in thepresence of a catalyst. The para hydrogen formed may be stored for longperiods, preferably at low temperature, e.g. 18-20K. Alternatively itmay be stored in pressurised gas form in containers which have an innersurface which is non-magnetic and non-paramagnetic.

The preparation of a para hydrogen-containing species is achieved byexposing an unsaturated precursor (containing NMR active nuclei) of thecompound to the para hydrogen-enriched hydrogen gas in the presence of asuitable catalyst. This enriched hydrogen will then react with theprecursor by reduction imparting a non-thermodynamic spin configurationto the target molecule. The compounds suitable for use are thus preparedfrom precursors which can be reduced by hydrogenation and which willtherefore typically possess one or more unsaturated bonds, e.g. doubleor triple carbon-carbon bonds.

When the p-H₂ molecule is transferred to the precursors of the compound(by means of catalytic hydrogenation with e.g. (PPh₂)₃RhCl), the protonspins remain anti parallel and begin to relax to thermal equilibriumwith the normal constant T1 of the hydrogen in the compound. However,during relaxation some of the polarisation may be transferred toneighbouring nuclei by pulse sequence Progress in Nuclear Spectroscopy,31, (1997), 293-315), low field cycling or other types of coupling. Thepresence of the NMR active nuclei as e.g. ¹³C (and ¹⁵N etc) with asuitable substitution pattern close to the relaxing hydrogen may lead tothe polarisation being trapped in the slowly relaxing ¹³C (and ¹⁵N etc)resulting in a high enhancement factor.

This embodiment is suitable for the aspects of the invention in whichthe NMR active nuclei in the test compound is hyperpolarised prior toadministration to the system in which its fate is to be tested.

A further hyperpolarisation transfer embodiment of this invention is thespin refrigeration method. This method covers spin polarisation of asolid compound or system by spin refrigeration polarisation. The systemis doped with or intimately mixed with a suitable paramagnetic materialsuch as Ni²⁺, lanthanide and actinide ions in crystal form with asymmetry axis of order three or more. The instrumentation is simplerthan that required for DNP, with no need for a uniform magnetic fieldsince no resonant excitation field is applied. The process is carriedout by physically rotating the sample around an axis perpendicular tothe direction of the magnetic field. The pre-requisite for this methodis that the paramagnetic species has a highly anisotropic g-factor. As aresult of the sample rotation, the electron paramagnetic resonance willbe brought in contact with the nuclear spins, leading to a decrease inthe nuclear spin temperature. Sample rotation is carried out until thenuclear spin polarisation has reached a new equilibrium. Again, it willbe appreciated that this embodiment is not suitable for polarising abiological system where that system is a whole animal, isolated organ ortissue or cultured cells.

When a test compound, system or sample from a system has beenhyperpolarised, it is desirable to preserve as much as possible of thepolarisation prior to NMR analysis. Some of the hyperpolarisationtechniques described above are only effective when transferringpolarisation in the solid state. However, it is often desired toinvestigate the NMR spectrum of a compound sample or system in theliquid state, in order to improve spectral resolution and sensitivity.Alternatively, line-narrowing techniques such as Magic Angle Spinning(MAS) can be employed to increase spectral resolution of NMR in thesolid state and enable low temperature NMR analysis.

If the compound, sample or system is not solid, it may conveniently befrozen in an appropriate solvent mixture prior to polarisation transferby one of the methods that needs to be carried out in the solid state.Solvent mixtures have been found to be particularly suitable, especiallyif the mix forms an amorphous glass. The amorphous matrix is employed toensure homogenous intimate mixing of radical and target in the solidwhile the sample is subject to DNP polarisation.

If a liquid state NMR technique is to be employed, once the compound,sample or system has been hyperpolarised, it can be rapidly removed fromthe polarisation chamber and then dissolved in a suitable solvent. It isadvantageous to use solvents that will not interfere with the images or,more usually, the spectra produced in the analysis step. Deuteratedsolvents such as D₂O are particularly suitable. Stirring, bubbling,sonification or other known techniques can be used to improve the speedof dissolution. Suitably, the temperature and pH of the solution aremaintained to allow optimal dissolution and a long nuclear relaxationtime.

Preferably, the compound, sample or system and then the solution thereofare kept in a holding field throughout the period between polalrisationand analysis in order to prevent relaxation. A holding field provides afield higher than the Earth's magnetic field and suitably higher than 10mT. It is suitably uniform in the region of the sample. Although aholding field is not required for all test compounds, much betterresults are obtained for some test compounds when such a field is usedand it is difficult to predict in advance which compounds will requiresuch a holding field, especially if the structure of the compound is notknown a priori. Therefore, it is preferable to use a holding fieldwhenever a system or sample is polarised and then transferred foranalysis. The optimal conditions will depend on the nature of thecompound, sample or system. The solution is subsequently transferred forexamination by standard solution phase NMR analysis. The transferprocess is preferably automated. Alternatively, the polarisationtransfer and dissolution steps are suitably integrated into a singleautomated unit. In an additional suitable embodiment, the polarisationtransfer and sample dissolution steps are automated and NMR detectionhardware is also housed within the same single fully integrated unit. Aholding field will not be required with such a fully integrated system.

Different ¹³C centres relax at very different rates. Consequently, verydifferent signal intensities would be expected to appear in theresulting NMR spectra if nuclear relaxation occurs during the transferfrom the hyperpolarisation unit to the NMR spectrometer. Surprisingly,peak heights from different centres within uniformly ¹³C enrichedmolecules have been observed to be of the same order. A possibleexplanation is that the effect may be due to a redistribution of theenhanced polarisation by cross-relaxation at certain carbon centres.This is useful, because it allows more information to be obtained thanmay otherwise have been expected. It is not unreasonable to assume thatany carbon centre within a given test compound will be detected withsimilar sensitivity.

Alternatively, where a solid state NMR technique is to be used, thesolid state compound, sample or system may be hyperpolarised, e.g. byDNP, brute force, spin refrigeration transfer or any other method thatwill work in the solid state at low temperature. Subsequently, thehyperpolarised sample will be moved into a solid-state MAS NMR probe.The movement is suitably rapid and is conveniently carried out vialifting or ejection. The sample in the NMR probe will then be spun sothat high-resolution solid state NMR spectroscopy can be carried out.The entire process can be automated and will preferably be carried outin an integrated unit.

The invention will now be illustrated by reference to the followingnon-limiting examples.

EXAMPLES

Trityl Radical

Stable triarylmethyl reagents are particularly suitable for DNPenhancements. The radical used in the following examples is tris(8-carboxyl-2,2,6,6,-tetra(2(1-hydroxyethyl))benzo[1,2-d:4,5-d′]bis(1,3)dithiol-4-yl)methylsodium salt (hereafter called trityl radical). This was made accordingto the methods described in WO98/39277.

Stock solutions of trityl radical were prepared in deuterated glycerolfor each example.

To glycerol-D₈ (200 μl) was added trityl radical (6.28 mg). The radicalwas dissolved by stirring under gentle heating and brief sonication andwas stored in a closed vial until required. This yielded a stock at 22mM which was used in the study of benzoic acid (example 1); the finaltrityl radical concentration used for the DNP step was 13.2 mM. Samplesof trityl radical solution were gently warmed to facilitate subsequentdispensing. Similarly a stock of trityl radical was prepared at 22.25 mMand this was employed for the hippuric acid in urine sample (example 4)with a final radical concentration of 14.9 mM for the DNP step.Additionally a stock was prepared at 25 mM and this was used for theremaining studies (examples 2,3,5 and 6), with final radicalconcentrations of 15 mM for the DNP steps.

40% ^(W)/v Sodium deutoxide was purchased from SIGMA/ALDRICH chemicalcompany and was diluted 100 fold in D₂O to prepare a 0.4% ^(W)/v. stockwhich was used in examples 1,2 and 3.

Glycerol-D₈, D₂O and DMSO-D₆ reagents were purchased from SIGMA/ALDRICH

Example 1 Study of (¹³C-carboxyl) Benzoic Acid Isolated From Rat Urine.

(¹³C-carboxyl) Benzoic acid was purchased from SIGMA/Aldrich chemicalcompany. A sample was added to rat urine at approximately 5 mg per mland then isolated by solid phase extraction (SPE) and reversed phasehigh performance liquid chromatography (RP hplc, C18 Kromasil, 25×1 cm,5 μm) using gradient elution with formic acid in water and formic acidin methanol. The isolated material was then dried and an aliquotre-analysed by RP hplc indicating a purity of 94%, with the maincomponent co-eluting with carrier material, monitored by on-line UVdetection (at 254 nm) and yielding the expected (M—H)⁻ ion at 122 massunits by on-line electrospray ionisation mass spectrometry (EI-MS).

A sample of (¹³C-carboxyl) benzoic acid (89 μg, 0.72 μmol) obtained asdescribed above was dissolved in sodium deutoxide in D₂O (0.4% ^(W)/v,7.5 ul, approximately 1.5 equivalents) and D₂O (7.5 μl). Trityl radicaldissolved in glycerol-D₈ (24 μl, 22 mM) was added to the benzoic acidsolution and the subsequent cocktail was mixed to homogeneity with adisposable plastic pipette. The sample was rapidly frozen, as smalldroplets, by dripping via a fine plastic pipette into a liquid nitrogenbath. The frozen sample drops were collected using small tweezers andplaced in a liquid nitrogen cooled Kel-F cup and this was transferredfor sample polarisation. The sample was polarised overnight at amagnetic field of 3.354T and at a microwave frequency of 93.925 GHz.Microwave power was 100 mW and sample temperature was maintained at1.25K for the duration of polarisation. The test sample was dissolved inhot D₂O (approximately 5 ml) in situ and a portion (approximately 1 mlin a 5 mm NMR tube, estimated sample temperature is 333K) was rapidlytransferred to an INOVA 400 MHz spectrometer for measurement of a liquidstate NMR spectrum. The sample was exposed to the earth's magnetic fieldduring transit to the spectrometer (approximately 20 seconds transfertime). The signal to noise estimated for the carboxyl carbon of(¹³C-carboxyl) benzoic acid peak, observed at 176.0 ppm is approximately440. The NMR spectrum was obtained in a single acquisition (acquisitiontime was 1.2 seconds, sweep width 25 kHz, following a RF pulse of 6microseconds, with WALTZ proton de-coupling applied during the pulse andacquisition; line broadening of 1 Hz was applied and signal to noise wasdetermined by Varian Vnmr software). The same sample was subsequentlyanalysed by non enhanced NMR with proton decoupling in the samespectrometer. A thermal equilibrium signal for the carbonyl carbon wasobtained with a signal to noise of approximately 8. This controlspectrum was acquired in 37 hours (averaging 168000 scans with a scanrepetition rate of 0.8 seconds and a flip angle of 11.2 degrees i.e.under Ernst angle conditions.

The sample used for NMR analysis was retained and subsequentlyre-analysed by RP hplc (C18 Kromasil, 25×0.46 cm, 5 μm) with gradientelution with formic acid in water and formic acid in acetonitrile and byEI-MS).

Re-analysis indicated predominantly (¹³C) benzoic acid and the materialyielded the expected molecular ion by MS analysis.

Results

The spectrum of FIG. 1 was obtained. The prominent peak from thelabelled ¹³COOH site of benzoic acid is clearly visible at 176.0 ppm.The prominent signals at around 71.9 ppm and around 62.3 ppm areidentified as solvent peaks from glycerol. The acquisition time for thisspectrum was a matter of seconds, compared to the days that would berequired by conventional NMR. The spectrum illustrates an exceptionalsignal to noise ratio.

Conclusions

The observed signal to noise ratio of the enhanced NMR spectrum,compared with the signal to noise ratio of the conventional NMR spectrumfrom the same sample, confirms that the method according to theinvention gives substantial improvement. The enhancement is estimated tobe of the order of a few thousandfold. Similarly, the remarkably shortdata acquisition time is evidence that the methods of the invention maybe used to carry out studies that would simply be too time-consuming todo in practice using conventional NMR.

The lines of the spectrum are very narrow and are positioned as expectedin the spectrum. Therefore, it can be inferred that the DNP radical isnot affecting the quality of the NMR spectrum. This is important becauseit shows that the signal enhancement obtained by using thishyperpolarisation method of the invention is not compromised byartefacts in the enhanced spectrum obtained.

Example 2 Study of (¹³C-carboxyl) Hippuric Acid (Primary Benzoic AcidMetabolite) Isolated from Rat Urine After iv Administration of(¹³C-carboxyl) Benzoic Acid.

Embodiment Without a Holding Field Magnet

(¹³C-carboxyl) Benzoic acid was administered intravenously at 10 mg/Kg(at 0 and 2 hrs) to 4 anaesthetised rats; urine was collected fromcanulated urethra. The major metabolite (¹³C-carboxy) hippuric acid wasisolated by SPE and RP-hplc. The isolated material was then dried and analiquot re-analysed by RP hplc indicating a purity of 99%, with the maincomponent co-eluting with authentic carrier material, monitored byon-line UV detection (at 254 nm) and yielding the expected (M−H)⁻ion at179 mass units by on-line electrospray ionisation mass spectrometry(EI-MS).

A sample of (¹³C-carboxyl) hippuric acid (463 μg, 2.57 μmol) obtained asdescribed above was dissolved in sodium deutoxide in D₂O (0.4% ^(W)/v,17.5 μl, approximately 0.97 equivalents). Trityl radical dissolved inglycerol-D₈ (26 μl, 25 mM) was added to the hippuric acid solution andthe subsequent cocktail was mixed to homogeneity with a disposableplastic pipette. The sample was rapidly frozen, as small droplets, bydripping via a fine plastic pipette into a liquid nitrogen bath. Thefrozen sample drops were collected using small tweezers and placed in aliquid nitrogen cooled Kel-F cup and this was transferred for samplepolarisation. The sample was polarised for 4 hours at a magnetic fieldof 3.354T and at a microwave frequency of 93.925 GHz. Microwave powerwas 100 mW and sample temperature was maintained at 1.25K for theduration of polarisation. The test sample was dissolved in hot D₂O (2-3ml) in situ and a portion (approximately 1 ml in a 5 mm NMR tube,estimated sample temperature is 333K) was rapidly transferred to anINOVA 400 MHz spectrometer for measurement of a liquid state NMRspectrum.

Results

The NMR spectrum was obtained in a single acquisition (acquisition timewas 1.2 seconds, sweep width 25 kHz, following a RF pulse of 6microseconds; ¹H decoupling was employed as above; line broadening of 1Hz was applied and signal to noise was determined by Varian Vnmrsoftware). A very noisy NMR spectrum from Hippuric acid was obtained.The signal to noise estimated for the carboxyl carbon peak of(¹³C-carboxyl) hippuric acid, observed at approximately 170 ppm is lessthan 4. Strong glycerol signals were observed.

Results

Example 3 Study of (¹³C-carboxyl) Hippuric Acid (Benzoic AcidMetabolite) Isolated from Rat Urine After iv Administration of(¹³C-carboxyl Benzoic Acid.

Embodiment Utilising a Holding Magnet

A sample of (¹³C-carboxyl) hippuric acid (463 μg, 2.57 μmol) obtained asdescribed above was dissolved sodium deutoxide in D₂O (0.4% ^(W)/v, 17.5μl, approximately 0.97 equivalents). Trityl radical dissolved inglycerol D₈ (26 μl, 25 mM) was added to the hippuric acid solution andthe subsequent cocktail was mixed to homogeneity with a disposableplastic pipette. The sample was rapidly frozen, as small droplets, bydripping via a fine plastic pipette into a liquid nitrogen bath. Thefrozen sample drops were collected using small tweezers and placed in aliquid nitrogen cooled Kel-F cup and this was transferred for samplepolarisation. The sample was polarised for 4 hours at a magnetic fieldof 3.354T and at a microwave frequency of 93.925 GHz. Microwave powerwas 100 mW and sample temperature was maintained at 1.25K for theduration of polarisation. The test sample was dissolved in hot D₂O (2-3ml) in situ and a portion (approximately 1 ml in a 5 mm NMR tube,estimated sample temperature is 333K) was rapidly transferred to anINOVA 400 MHz spectrometer for measurement of a liquid state ¹³C NMRspectrum. The sample was maintained in a magnetic holding field of 10 mTduring transit to the spectrometer (approximately 20 seconds transfertime). The signal to noise estimated for the carboxyl carbon of(¹³C-carboxyl) hippuric acid peak, observed at 171.2 ppm, isapproximately 1500. An NMR spectrum was obtained in a single acquisition(acquisition time was 1.2 seconds, sweep width 25 kHz, following a RFpulse of 6 microseconds; ¹H decoupling was employed as above linebroadening of 1 Hz was applied and signal to noise was determined byVarian Vnmr software).

The sample used for NMR analysis was retained and subsequentlyre-analysed by RP hplc (C18 Kromasil, 25×0.46 cm, 5 μm) with gradientelution with formic acid in water and formic acid in acetonitrile and byEI-MS). Re-analysis indicated predominantly (¹³C) hippuric acid and thematerial yielded the expected molecular ion by MS analysis.

Results

The enhanced NMR spectrum is shown in FIG. 2. A much higher signal tonoise ratio for the hippuric acid NMR signal was obtained in thisexample using a holding field than in Example 2 where no holding fieldwas used.

Pleasingly, peaks in addition to that expected for the label wereobserved.

The chemical shift difference of the NMR signal from the hippuric acidlabel compared to that from the benzoic acid label was as expected fromconventional NMR studies.

Conclusions

It is beneficial to use a holding field when analysing some compounds bya technique where the sample is polarised in one location and NMRanalysis takes place in another. Since it is not always possible topredict in advance which compounds will benefit from the use of such aholding field, it should be used routinely when ex situhyperpolarisation techniques are employed.

The presence of additional peaks from the hippuric acid in the enhancedNMR spectrum indicates that the methods of the invention are suitablefor studying test compounds at lower doses than would be possible usingthe conventional NMR. This is potentially significant for studyingtoxicity and or metabolism of drugs and drug candidates, where somemetabolites may be present only at very low concentrations.

Moreover, it can be inferred from the presence of additional peaks thatthe methods of the invention may be suitable for use with test compoundswherein there is a lower degree of enrichment or even natural abundanceof the NMR active nuclei.

The observation of NMR signals at the expected chemical shift positionsconfirms that this method of the invention does not introduce artefactsand can be used to study the fate of the test compound.

Example 4 (¹³C-carboxyl) Hippuric Acid (Analysed Directly After Spikinginto Rat Urine)

(¹³C-carboxyl) Hippuric acid was synthesised in-house from(¹³C-carboxyl) benzoic acid and glycine.

A sample was added to rat urine at approximately 5 mg/ml, which was thelevel measured in urine (0-2 hr collection) in the benzoic acidmetabolism study described above. A sample containing 95 μg of synthetic(¹³C-carboxyl) hippuric acid was dissolved in approximately 20 μl of raturine. Trityl radical dissolved in glycerol-D₈ (30 ul, 22.25 mM) wasadded to the hippuric acid solution and the subsequent cocktail wasmixed to homogeneity with a disposable plastic pipette. The sample wasrapidly frozen, as small droplets, by dripping via a fine plasticpipette into a liquid nitrogen bath. The frozen sample drops werecollected using small tweezers and placed in a liquid nitrogen cooledKel-F cup and this was transferred for sample polarisation. The samplewas polarised for 4 hours at a magnetic field of 3.354T and at amicrowave frequency of 93.925 GHz. Microwave power was 100 mW and sampletemperature was maintained at 1.25K for the duration of polarisation.The test sample was dissolved in hot D₂O (2-3 ml) in situ and a portion(approximately 1 ml in a 5 mm NMR tube, estimated sample temperature is333K) was rapidly transferred to an INOVA 400 MHz spectrometer formeasurement of a liquid state ¹³C NMR spectrum. The sample wasmaintained in a magnetic holding field of 10 mT during transit to thespectrometer (approximately 20 seconds transfer time).

The signal to noise estimated for the carboxyl carbon of (¹³C-carboxyl)hippuric acid peak, observed at 171.1 ppm, is approximately 534. An NMRspectrum was obtained in a single acquisition (acquisition time was 1.2seconds, sweep width 25 kHz, following a RF pulse of 6 microseconds ¹Hdecoupling was employed as above; line broadening of 1 Hz was appliedand signal to noise was determined by Varan Vnmr software).

The sample used for NMR analysis was retained and subsequentlyre-analysed by RP hplc (C18 Kromasil, 25×0.46 cm, 5 um) with gradientelution with formic acid in water and formic acid in acetonitrile and byEI-MS). Re-analysis indicated predominantly (¹³C) hippuric acid and thematerial yielded the expected molecular ion by MS analysis.

Results

Two NMR signals were observed, one arising from hippuric acid at 170.7ppm and one smaller singlet NMR signal at 163.1 ppm that can be assignedtentatively to urea, because its chemical shift is consistent with theshift predicted using ACD Labs software, and this substance is known tobe a major constituent of urine.

Conclusions

In this case, a test compound was analysed directly in a biologicalmatrix, i.e. rat urine. The fact that a clear signal was obtained isvery encouraging and confirms that samples collected over time could beanalysed, thus enabling dynamic studies. Another conclusion is thatsamples may not need to be fractionated prior to NMR analysis.

It is not unreasonable to infer that had benzoic acid also been presentin the sample, it would also have been detected. Accordingly,pharmacokinetic studies could be undertaken using the methods of theinvention.

Example 5 (U-¹³C)Paracetamol Isolated from Rat Urine

(U-¹³C)Paracetamol was synthesised. This material was added to rat urineat approximately 5 mg per ml and then isolated by solid phase extraction(SPE) and reversed phase high performance liquid chromatography (RPhplc, C18 Kromasil, 25×1 cm, 5 μm) using gradient elution with formicacid in water and formic acid in methanol.

The isolated material was then dried and an aliquot reanalysed by RPhplc indicating a purity of 94%, with the main component co-eluting withcarrier material monitored by on-line UV detection (at 254 nm) andyielding the expected (M+H)⁺ ion at 160 mass units by on-lineelectrospray ionisation mass spectrometry (EI-MS).

A sample of (U-¹³C) paracetamol (312 μg, 1.96 μmol) obtained asdescribed above was dissolved in DMSO-D₆ (10 μl) and D₂O (14 μl). Tritylradical dissolved in glycerol-D₈ (36 μl, 25 mM) was added to the (U-¹³C)paracetamol solution and the subsequent cocktail was mixed tohomogeneity with a disposable plastic pipette. The sample was rapidlyfrozen, as small droplets, by dripping via a fine plastic pipette into aliquid nitrogen bath. The frozen sample drops were collected using smalltweezers and placed in a liquid nitrogen cooled Kel-F cup and this wastransferred for sample polarisation. The sample was polarised for 4hours at a magnetic field of 3.354T and at a microwave frequency of93.925 GHz. Microwave power was 100 mW and sample temperature wasmaintained at 1.25K for the duration of polarisation. The test samplewas dissolved in hot D₂O (2-3 ml) in situ and a portion (approximately 1ml in a 5 mm NMR tube, estimated sample temperature is 333K) was rapidlytransferred to an INOVA 400 MHz spectrometer for measurement of a liquidstate ¹³C NMR spectrum. The sample was maintained in a magnetic holdingfield of 10 mT during transit to the spectrometer (approximately 20seconds transfer time). An NMR spectrum was acquired in a singleacquisition (acquisition time was 1.2 seconds, ¹H discoupling wasemployed as above, sweep width 25 kHz, following a RF pulse of 6microseconds).

The sample used for NMR analysis was retained and subsequentlyre-analysed by RP hplc (C18 Kromasil, 25×0.46 cm, 5 um) with gradientelution with formic acid in water and formic acid in acetonitrile and byEI-MS). Re-analysis indicated predominantly (¹³C) paracetamol and thematerial yielded the expected molecular ion by MS analysis.

Results

An enhancement was observed with all peaks being present at positionsconsistent with prediction.

Example 6 Study of (U-¹³C)paracetamol-sulphate Isolated from Rat BileAfter iv Administration of (U-¹³C)paracetamol

(U-¹³C)Paracetamol was administered intravenously at 20 mg/kg (at 0 and3 hrs) to 4 anaesthetised rats, urine was collected from canulatedurethra and bile was collected from canulated bile duct (2 rats only) at0-3 and 3-6 hours. Bile (3-6 hrs) was extracted with dichloromethane andthen fractionated by RP-hplc (C18 Kromasil, 25×1 cm, 5 um) usinggradient elution with formic acid in water and formic acid in methanol).

Several metabolites were collected. Paracetamol sulphate was identifiedby on-line EI-MS, from its (M+H)⁺ ion at 240 mass units. The isolatedmaterial was then dried and an aliquot re-analysed by RP hplc indicatinga peak purity of 96.7%, monitored by on-line UV detection (at 254 nm)and gave the expected (M+H)+ ion at 240 mass units by on-lineelectrospray ionisation mass spectrometry (EI-MS).

A sample of (U-¹³C) paracetamol-sulphate (100 g, 0.42 μmol) obtained asdescribed above was dissolved in D₂O (24 μl). Trityl radical dissolvedin glycerol-D₈ (36 μl, 25 mM) was added to the (U-¹³C) paracetamolsolution and the subsequent cocktail was mixed to homogeneity with adisposable plastic pipette. The sample was rapidly frozen, as smalldroplets, by dripping via a fine plastic pipette into a liquid nitrogenbath. The frozen sample drops were collected using small tweezers andplaced in a liquid nitrogen cooled Kel-F cup and this was transferredfor sample polarisation. The sample was polarised for 4 hours at amagnetic field of 3.354T and at a microwave frequency of 93.925 GHz.Microwave power was 100 mW and sample temperature was maintained at1.25K for the duration of polarisation. The test sample was dissolved inhot D₂O (2-3 ml) in situ and a portion (approximately 1 ml in a 5 mm NMRtube, estimated sample temperature is 333K) was rapidly transferred toan INOVA 400 MHz spectrometer for measurement of a liquid state ¹³C NMRspectrum. The sample was maintained in a magnetic holding field of 10 mTduring transit to the spectrometer (approximately 20 seconds transfertime). An NMR spectrum was acquired in a single acquisition (acquisitiontime was 1.2 seconds, sweep width 25 kHz, following a RF pulse of 6microseconds ¹H decoupling was applied as above).

The sample used for NMR was retained and subsequently re-analysed by RPhplc (C18 Kromasil, 25×0.46 cm, 5 μm) with gradient elution with formicacid in water and formic acid in acetonitrile and by EI-MS). Re-analysisindicated a major component with a retention time consistent withparacetamol sulphate from previous analysis and the material yielded theexpected molecular ion by MS analysis.

Results

The enhanced NMR spectrum is shown in FIG. 3. Differences were observedin the spectrum for paracetamol sulphate compared with paracetamol. Inparticular, the chemical shift positions for ¹³C NMR signals arisingfrom aromatic sites are significantly different.

The peak heights for the carbon centres were of the same order.

Conclusions

The paracetamol sulphate had been produced by metabolism in a rat andwas collected from bile. Bile is a different biological matrix thanurine (from which the hippuric acid and benzoic acid were collected. Theresults indicate that NMR enhancements can be observed irrespective ofthe biological matrix from which the test compound is derived. The factthat a different spectrum was obtained compared with paracetamolconfirms that it may be possible to differentiate between peaks fromparent compounds and their metabolites in a mixture, even in the case ofsubtle structural differences between them.

A pleasing observation was that, as in all the other examples, nosignals arising from the radical, were observed.

It was surprising to see that the peak heights for the carbon centreswere of the same order. Since different carbon centres decay at verydifferent rates, it was expected that the signal intensities for thecarbon centres would be very different. Indeed, it was not expected tobe possible to observe the methyl peaks at all. Therefore, moreinformation was obtained from the experiment than anticipated. It is notunreasonable to assume that any carbon centre within a given testcompound would be detected with similar sensitivity by the method of theinvention.

1. A method for investigating the fate of a test compound, said methodcomprising the steps of: administering the test compound to a biologicalsystem in which the fate of the test compound is to be studied, whereinthe test compound is enriched with ¹³C; extracting samples from thesystem over time; hyperpolarising the NMR active nuclei in the samplesextracted from the system over time, wherein said hyperpolarising iscarried out by polarization transfer using solid state dynamic nuclearpolarization (DNP) effected by a DNP agent; subjecting the samples to atreatment resulting in solid to liquid phase transition; and analysingeach of said samples by NMR spectroscopy in the liquid state.
 2. Amethod according to claim 1, wherein the hyperpolarised sample isretained in a holding field in the period from hyperpolarisation toanalysis.
 3. A method according to claim 1, wherein the metabolism of atest compound is studied.
 4. A method according to claim 1, wherein thesystem is one of a whole animal and a human body.
 5. A method accordingto claim 1 for studying absorption of a test compound wherein the systemis one of a whole animal and a human body.